The genetic stability of an organism depends on the accurate partition of sister chromatids into two daughter cells during mitosis, which in turn requires the maintenance of the physical linkage (cohesion) between sister chromatids until their bipolar attachment to the mitotic spindle. Removal of cohesin (theprotein complex that maintains sister chromatid cohesion) from chromosomes then leads to sister chromatid separation. In vertebrate cells, removal of cohesin occurs in two steps. At prophase, most of cohesin along the chromosome arms is removed through Polo-like kinase (Plk1)/Aurora B-dependent phosphorylation of cohesin. At metaphase, the residual centromeric pool of cohesin is cleaved by separase to allow sister chromatid separation. The long-term goal of my lab is to understand the molecular mechanism of chromosome segregation in mammalian cells. In this proposal, we will address an interesting puzzle in this area: how is the centromeric cohesin shielded from the actions of Plk1 /Aurora B in prophase? We have recently provided evidence to suggest that the spindle checkpoint kinase Bub1 targets and the Sgo1 centromeric protein and protein phosphatase 2A (PP2A) to centromeres where they counteract the phosphorylation of cohesin by Plk1and other mitotic kinases. In Specific Aims 1 and 2, we will further delineate the mechanisms by which Bub1, Sgo1, and PP2A collaborate to protect centromeric cohesion. On the other hand, our results also point to a PP2A-independent role of Sgo1. Indeed, we have made two novel and related findings in the past year. Sgo1 directly interacts with an RNA-binding protein complex called ILF2-ILF3 (interleukin enhancer binding factors 2 and 3). Sgo1 itself binds to RNA in vitro. Experiments are planned in Aims 3 and 4 to establish the in vivo relevance of these findings. Premature loss of centromeric sister chromatid cohesion leads to chromosome missegregation and abnormal numbers of chromosomes in daughter cells (aneuploidy), which contributes to cancer formation and birth defects. The proposed research will shed light on the mechanism of chromosome segregation and may in turn lead to better understanding and prevention of chromosomal instability and aneuploidy in human cancers and birth defects, such as Down Syndrome.